Test piece for protein assay and process for producing the same

ABSTRACT

The present invention relates to a test piece for protein assay that is used for quantifying or semi-quantifying a protein and that contains an acidic pH indicator. This test piece for protein assay contains a surfactant as a sensitizer for increasing coloration sensitivity with respect to protein. A triphenylmethane-based indicator is used as the acidic pH indicator, for example. The present invention also provides a method for manufacturing a test piece for protein assay. With this method, a test piece for protein assay is manufactured by impregnating an absorbent carrier with an impregnant containing an acidic pH indicator and a sensitizer, and then drying this product. A surfactant is used as the sensitizer.

TECHNICAL FIELD

This invention relates to a test piece for protein assay for assayingproteins present in protein-containing samples (body fluid such as bloodand urine, or protein-containing beverages, or factory wastewater,etc.), and also relates to a method for manufacturing such a test piece.

BACKGROUND ART

Assaying the protein in a biological sample is important in pathologicaldiagnosis. For instance, the amount of serum albumin decreases in thecase of diminished liver function, while the amount of protein in urineincreases in the case of nephritis, nephrotic syndrome, lithiasis,tumors, other such kidney and urinary tract disorders, disorders of thecirculatory system and disorders of central nervous system. Therefore,assaying albumin or other proteins can be an important clue in thediagnosis of these disorders.

A simple assay method featuring the use of a protein error indicator isknown in the field of protein assay. With this assay method,tetrabromophenol blue (TBPB) is used, for instance, as the protein errorindicator. As one example, urine test paper made with TBPB is widelyused for primary screening purposes. TBPB changes from yellow to bluethrough the dissociation of phenolic hydroxyl groups at a pH of about 3when a protein is present, and therefore can be used to detect protein.

However, test paper made using TBPB as the indicator has inadequatesensitivity with respect to the low protein concentrations of 10 to 20mg/dL required for clinical use, and is therefore sometimes incapable ofdetecting protein accurately. For example, in a visual evaluationconducted by comparison with a color chart, the color is very similarbetween negative protein and trace protein, making it difficult to tellthe two apart and hampering accurate evaluation. Meanwhile, when a urinetest paper assay apparatus is used, the low sensitivity of TBPB oftenresults in erroneous evaluation.

Consequently, there has been a need for a technique that would allow lowconcentrations of protein to be quantified at higher sensitivity.

DISCLOSURE OF THE INVENTION

As a result of diligent research aimed at solving the above problems,the inventors arrived at the present invention upon discovering that lowconcentrations of protein can be assayed at high sensitivity if the testpiece contains a surfactant.

Specifically, the test piece for protein assay provided by a firstaspect of the present invention is a test piece for protein assay thatis used for quantifying or semi-quantifying a protein and that containsan acidic pH indicator, containing a surfactant as a sensitizer forincreasing coloration sensitivity to protein.

A second aspect of the present invention is a method for manufacturing atest piece for protein assay that is used for quantifying orsemi-quantifying a protein, by impregnating an absorbent carrier with animpregnant containing an acidic pH indicator and a sensitizer, and thendrying this product, wherein a surfactant is used as the sensitizer.

An example of an acidic pH indicator that can be used in the presentinvention is a triphenylmethane-based indicator. A typical example of atriphenylmethane-based indicator is expressed by the following ChemicalFormula (1).

In Chemical Formula (1), X1 is a halogen, a nitro group, or a nitrosogroup, and X2 and X3 are the same or different halogens.

With the present invention, it is preferable to use the tetrabromophenolblue (TBPB) expressed by the following Chemical Formula (2) as thetriphenylmethane-based indicator.

There are no particular restrictions on the concentration of the acidicpH indicator (such as TBPB) in the impregnant, but it is typically 0.1to 5 mM, and preferably 0.3 to 1 mM.

It is preferable to use a cationic surfactant as the surfactant(sensitizer). In addition to this, or instead of this, a nonionicsurfactant may be used.

A quaternary ammonium salt can be used, for example, as the cationicsurfactant. This quaternary ammonium salt can be analkyldimethylbenzylammonium, alkyltrimethylammonium salt,dialkyldimethylammonium salt, benzalkonium salt, imidazolium salt, orthe like. An aliphatic amine salt can also be used as the cationicsurfactant.

The nonionic surfactant can be either an ether type, ether ester type,ester type, or nitrogen-containing type.

Examples of ether-type surfactants include polyoxyethylene alkyl ether,polyoxyethylene secondary alcohol ether, polyoxyethylene alkylphenylether, polyoxyethylene sterol ether, polyoxyethylene lanolin derivative,ethylene oxide derivative of alkylphenol formalin condensate,polyoxyethylene polyoxypropylene block polymer, and polyoxyethylenepolyoxypropylene alkyl ether.

Examples of ether ester-type surfactants include polyoxyethyleneglycerol fatty acid ester, polyoxyethylene castor oil, polyoxyethylenehydrogenated castor oil, polyoxyethylene sorbitan fatty acid ester, andpolyoxyethylene sorbitol fatty acid ester.

Examples of ester-type surfactants include polyethylene glycol fattyacid ester, fatty acid monoglyceride, monoglycerol fatty acid ester,sorbitan fatty acid ester, propylene glycol fatty acid ester, andsucrose fatty acid ester.

Examples of nitrogen-containing surfactants include fatty acidalkanolamide, polyoxyethylene fatty acid amide,polyoxyethylenealkylamine, and alkylamine oxide.

The cationic surfactant used in the present invention is typicallybenzyltrimethylammonium bromide, hexadecyltrimethylammonium bromide,lauryltrimethylammonium bromide, or zephiramine, and the nonionicsurfactant is typically polyethylene glycol. Naturally, a plurality ofdifferent surfactants may be used together, in which case it ispreferable to use a combination of a cationic surfactant and a nonionicsurfactant. Typically, a combination of benzyltrimethylammonium bromide,which is a cationic surfactant, and polyethylene glycol, which is anonionic surfactant, is used.

There are no particular restrictions on the concentration of thesensitizer (surfactant) in the impregnant, but it is typically 0.01 to 5wt %, and preferably 0.01 to 1 wt %.

A polycarbonate, polyvinyl alcohol, or other such polymer material mayalso be used in addition to the surfactant as a sensitizer.

The pH of the impregnant is set to be somewhat lower than the pKa of theacidic pH indicator. When TBPB is used as the acidic pH indicator, thepH of the impregnant is to be from 2.0 to 4.5, and preferably 2.0 to3.5.

Any buffer can be used as long as it has a good buffering action at thepH of the impregnant (between 2.0 and 4.5, for instance) and does notimpede the reaction between the acidic pH indicator and the protein.Examples of buffers that can be used include glycine buffer, citratebuffer, succinate buffer, malate buffer, and tartrate buffer. There areno particular restrictions on the concentration of the buffer in theimpregnant, but it is typically from 0.1 to 1.5 M, and preferably 0.3 to1 M.

A porous substance that contains no protein component can be used as theabsorbent carrier, and can be used in the form of a sheet or film, forexample. Examples of porous substances include paper-like materials,foams, woven materials, nonwoven materials, and knits. Examples of thematerial used to form the absorbent carrier include cotton, linen,cellulose, nitrocellulose, cellulose acetate, rock wool, glass fiber,silica fiber, carbon fiber, boron fiber, polyamide, aramid, polyvinylalcohol, polyvinyl acetate, rayon, polyester, nylon, polyacrylic acid,polyacrylic ester, and polyolefin. There are no particular restrictionson the shape of the absorbent carrier, but it is generally rectangular(either short and wide or long and narrow), circular, or oval.

The test piece for protein assay of the present invention can be useddirectly as it is, or after first being bonded to a non-absorbentmaterial.

The non-absorbent material is used in the form of a sheet or film, forexample. Examples of the material used to form this non-absorbentmaterial include polyethylene terephthalate, polyester, polypropylene,polyethylene, polyvinyl chloride, polyvinylidene chloride, andpolystyrene.

EXAMPLES Example 1

In this example, each test piece was impregnated with urine having analbumin concentration of 0.3 mg/dL (negative) or 15 mg/dL (positive),and the reflectance of the test piece was measured. The test pieces wereformed by impregnating filter paper (3MMChr made by Whatman) with animpregnant, and then drying. The impregnant was produced by adding 0.2wt % benzyltrimethylammonium bromide (a cationic surfactant) as asensitizer to the base composition shown in Table 1. Reflectance wasmeasured with a calorimeter at a measurement wavelength of 630 nm. Themeasurement results are given in Table 2.

Example 2

In this example, reflectance was measured in the same manner as inExample 1, except that the impregnant was produced by addinghexadecyltrimethylammonium bromide (a cationic surfactant) as asensitizer in an amount of 0.01 wt % to the base composition in Table 1.The measurement results are given in Table 2.

Example 3

In this example, reflectance was measured in the same manner as inExample 1, except that the impregnant was produced by addinglauryltrimethylammonium bromide (a cationic surfactant) as a sensitizerin an amount of 0.01 wt % to the base composition in Table 1. Themeasurement results are given in Table 2.

Example 4

In this example, reflectance was measured in the same manner as inExample 1, except that the impregnant was produced by addingpolyethylene glycol (a nonionic surfactant) as a sensitizer in an amountof 0.5 wt % to the base composition in Table 1. The measurement resultsare given in Table 2.

Example 5

In this example, reflectance was measured in the same manner as inExample 1, except that the impregnant was produced by addingbenzyltrimethylammonium bromide (a cationic surfactant) in an amount of0.2 wt % and polyethylene glycol (a nonionic surfactant) in an amount of0.5 wt % as sensitizers to the base composition in Table 1. Themeasurement results are given in Table 2.

Example 6

In this example, reflectance was measured in the same manner as inExample 1, except that the impregnant was produced by addinglauryltrimethylammonium bromide (a cationic surfactant) in an amount of0.01 wt % and polyethylene glycol (a nonionic surfactant) in an amountof 0.5 wt % as sensitizers to the base composition in Table 1. Themeasurement results are given in Table 2.

Example 7

In this example, reflectance was measured in the same manner as inExample 1, except that the impregnant was produced by adding zephiramine(a cationic surfactant) in an amount of 0.01 wt % and polyethyleneglycol (a nonionic surfactant) in an amount of 0.5 wt % as sensitizersto the base composition in Table 1. The measurement results are given inTable 2.

Comparative Example 1

In this comparative example, reflectance was measured in the same manneras in Example 1, except that the impregnant was the base compositionitself (containing no surfactant (sensitizer)). The measurement resultsare given in Table 2. TABLE 1 Base composition Indicator Buffer (pH 3.4)Solvent TBPB malate buffer ethanol 0.5 mM 0.8 M 30 wt %

TABLE 2 Measurement results Reflectance (%) Differ- 0.3 mg/dL 15 mg/dLential Sensitizer (negative) (positive) Δ(%) Ex. 1benzyltrimethylammonium 60.7 39.3 21.4 bromide 0.2 wt % Ex. 2hexadecyltrimethylammonium 60.4 40.6 19.8 bromide 0.01 wt % Ex. 3lauryltrimethylammonium 59.0 38.7 20.3 bromide 0.01 wt % Ex. 4polyethylene glycol 57.0 40.4 16.6 0.5 wt % Ex. 5benzyltrimethylammonium 60.6 38.3 22.3 bromide 0.2 wt % polyethyleneglycol 0.5 wt % Ex. 6 lauryltrimethylammonium 59.1 35.9 23.2 bromide0.01 wt % polyethylene glycol 0.5 wt % Ex. 7 zephiramine 0.01 wt % 61.241.5 19.8 polyethylene glycol 0.5 wt % C. E. 1 none 60.7 48.9 11.8

As is clear from Table 2, reflectance at an albumin concentration of 15mg/dL was lower when a surfactant was contained than when no surfactantwas contained. Accordingly, the differential Δ (%) between negative (0.3mg/dL) and positive (15 mg/dL) when a surfactant was contained was abouttwice that when no surfactant was contained. We can therefore concludethat when a surfactant is contained, sensitivity to albumin is higher,and albumin can be properly detected even when the albumin concentrationis low (about 10 to 20 mg/dL). The albumin detection sensitivity wasparticularly high when a cationic surfactant was used alone and when itis used together with a nonionic surfactant. In this respect, a cationicsurfactant can be considered to be preferable as a sensitizer.

The experiment results in these examples pertain to urine samples, butthe present invention is not limited to urine, and can also be appliedto the quantification of protein in any of various other samplescontaining protein, such as blood, protein-containing beverages, andfactory wastewater.

1. A test piece for protein assay, said test piece used for quantifyingor semi-quantifying a protein and containing an acidic pH indicator,wherein said test piece contains a surfactant as a sensitizer forincreasing coloration sensitivity with respect to the protein.
 2. Thetest piece for protein assay according to claim 1, wherein the acidic pHindicator is a triphenylmethane-based indicator.
 3. The test piece forprotein assay according to claim 2, wherein the triphenylmethane-basedindicator is tetrabromophenol blue expressed by the following ChemicalFormula (1).


4. The test piece for protein assay according to claim 1, containing acationic surfactant as the sensitizer.
 5. The test piece for proteinassay according to claim 4, wherein the cationic surfactant is at leastone type selected from the group consisting of benzyltrimethylammoniumbromide, hexadecyltrimethylammonium bromide, lauryltrimethylammoniumbromide, and zephiramine.
 6. The test piece for protein assay accordingto claim 1, containing a nonionic surfactant as the sensitizer.
 7. Thetest piece for protein assay according to claim 6, wherein the nonionicsurfactant is polyethylene glycol.
 8. The test piece for protein assayaccording to claim 1, wherein a combination of the cationic surfactantand nonionic surfactant is used as the sensitizer.
 9. The test piece forprotein assay according to claim 8, wherein the cationic surfactant isbenzyltrimethylammonium bromide and the nonionic surfactant ispolyethylene glycol.
 10. A method for manufacturing a test piece forprotein assay that is used for quantifying or semi-quantifying aprotein, the method comprising the steps of impregnating an absorbentcarrier with an impregnant containing an acidic pH indicator and asensitizer, and drying the carrier, wherein a surfactant is used as thesensitizer.
 11. The method for manufacturing a test piece for proteinassay according to claim 10, wherein the surfactant is at least one typeselected from the group consisting of benzyltrimethylammonium bromide,hexadecyltrimethylammonium bromide, lauryltrimethylammonium bromide,zephiramine, and polyethylene glycol.
 12. The method for manufacturing atest piece for protein assay according to claim 11, wherein acombination of benzyltrimethylammonium bromide and polyethylene glycolis used as the surfactant.
 13. The method for manufacturing a test piecefor protein assay according to claim 10, wherein the amount ofsurfactant contained in the impregnant is set between 0.01 and 5 wt %.14. The method for manufacturing a test piece for protein assayaccording to claim 13, wherein the amount of surfactant contained in theimpregnant is set between 0.01 and 1 wt %.
 15. The method formanufacturing a test piece for protein assay according to claim 10,wherein the tetrabromophenol blue expressed by the following ChemicalFormula (2) is used as the acidic pH indicator.


16. The method for manufacturing a test piece for protein assayaccording to claim 10, wherein the concentration of the acidic pHindicator in the impregnant is set between 0.1 and 5 mM.
 17. The methodfor manufacturing a test piece for protein assay according to claim 16,wherein the pH of the impregnant is set at or below the pKa of theacidic pH indicator.
 18. The method for manufacturing a test piece forprotein assay according to claim 17, wherein the pH of the impregnant isset between 2.0 and 4.5.